# Patent

## Title
Inhibitors of hepatitis C virus

## Abstract

The present invention relates to methods that employ peptides or peptide derivatives to inhibit hepatitis C virus infection. The present invention is based in part on the discovery that E2 envelope glycoprotein of hepatitis C virus has previously undescribed domains that are important for interactions with cellular or viral proteins that are necessary for early steps in HCV infection. The present invention provides peptides and methods of treatment and prophylaxis of diseases induced by hepatitis C virus and related viruses.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. provisional patent application Ser. No. 60/614,280, entitled “Inhibitors of Hepatitis C Virus,” by Robert F. Garry, Jr. and Jane A. McKeating, filed Sep. 29, 2004, which is incorporated by reference herein in its entirety.


FIELD OF THE INVENTION
The present invention relates to peptides and methods of inhibiting virus-cell binding and entry of hepatitis C virus. Specific embodiments of the invention are drawn to the inhibition of infection by hepatitis C virus (HCV).
BACKGROUND OF THE INVENTION
Viruses must infect host cells to replicate, produce a spreading infection, and cause disease. Infection by enveloped viruses requires binding of the virion to one or more structures on the cell surface (Flint and McKeating, 2000). The initial step may be a low affinity non-specific binding (Barth et al., 2003). Subsequently, the virus binds with high affinity to primary receptors, and then in some cases to secondary receptors or co-receptors (Bartosch et al., 2003; Hsu et al., 2003; Roccasecca et al., 2003; Cormier et al., 2003;_Pohlmann et al., 2003; Zhang et al., 2004). The cell surface binding steps can be associated with a variety of structural rearrangements of the virion surface proteins and changes in protein-protein interactions between the viral surface proteins (Jardetsky and Lamb, 2004; Modis et al., 2004; Bressanelli et al., 2004; Gibbons et al, 2004). The latter steps can expose the fusion peptide, a hydrophobic domain of a viral glycoprotein that is able to interact with cell membranes (Flint et al., 1999; Allison et al., 2001). In some cases, the binding of the virus to the cell surface receptors triggers uptake of the virus through endocytic, or similar vesicular pathways (Garry and Dash, 2003; Jardetsky and Lamb, 2004). Exposure to more acidic conditions in the vesicles can trigger conformational changes in the viral surface proteins, including those that expose the fusion peptide (Kuhn et al., 2002; Lescar et al., 2001). For most viruses, binding to the cellular receptor is primarily the function of one viral surface protein, whereas fusion of the viral and cellular membranes is primarily the function of another viral surface protein. An example of a virus with separate receptor binding and fusion protein is HIV. The receptor binding protein of HIV is the surface glycoprotein (SU; gp120) and the fusion protein is the transmembrane glycoprotein (TM;gp41) (Kwong et al., 1998; Gallaher et al., 1987; 1989). Most viruses with class I fusion proteins in which the fusion peptide is located at or near the amino terminus, for example retroviruses, orthomyxoviruses, paramyxoviruses arenaviruses and coronaviruses, use one protein for receptor binding and another for fusion (Wilson et al., 1981; Gallaher et al, 1996; 2001). Alphaviruses, which have a class II fusion protein with an internal fusion peptide, also use one protein principally for receptor binding and another for fusion of the viral and cellular membranes (Straus and Straus, 1994). The envelope (E) protein encoded by members of the flavivirus genus of the Flaviviridae, has an internal fusion peptide, but serves both receptor binding and fusion function (Allison et al., 2001).
Hepatitis C virus encodes two envelope glycoproteins, E1 (gp35) and E2 (gp70), both with C-terminal transmembrane anchor domains (Flint and McKeating, 2000). E2 interacts with several cell surface proteins (CD81, SR-BI and L-SIGN) suggesting that it is the receptor binding protein of HCV (McKeating, 2004). The function of E1 is less clear and may act to chaperone E2 (Flint et al., 1999; Garry and Dash, 2003). Synthetic peptides corresponding to hepatitis C virus E2 can block infection mediated by hepatitis C virus. Structural determinations of the hepatitis C virus E2 allow the identification of several heretofore unknown features of hepatitis C virus E2 for drug and vaccine development.
The Flavivirus family includes a variety of important human and animal pathogens. Hepatitis C virus (HCV) is the leading viral cause of chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (Poynard et al., 2003). In the United States alone, an estimated 4 million people are infected with HCV. This is approximately four times the number infected by HIV. Each year in the US, 30-50,000 new HCV infections occur, and about 15-20,000 people die. Moreover, these numbers are expected to increase dramatically given that a substantial portion of HCV infected individuals show little or no response to the only currently approved therapeutics (i.e. treatment with interferons and/or ribavirin). HCV infection is spread primarily through needle sharing among drug users, although there is some risk from accidental needle sticks, blood products before 1992, chronic blood dialysis, and frequent sexual contact. Current treatments for HCV using ribavirin and interferon cost  ̃$8,000 to $20,000 per year, and are only partially successful in about half of patients treated. Overall, about 80% of HCV carriers suffer chronic liver inflammation and cirrhosis, of these 25% will develop end stage liver disease or hepatocellular carcinoma (HCC) (Colombo, 2000). End stage HCV disease is the most frequent indication for liver transplants and this costs $250,000 to $300,000. Better drugs to treat HCV infection and an effective vaccine to prevent HCV infection are urgently needed.
SUMMARY OF THE INVENTION
The present invention relates to the compositions comprising peptides or peptide derivatives and methods that employ these compositions to treat, prevent or inhibit infection by hepatitis C virus (HCV) and related viruses. The present invention is made possible by the inventors' discovery that that HCV encoded E2 glycoprotein (and the analog(s) from related viruses) has previously undescribed domains that are important for interaction(s) and rearrangements of E2 with E2 and/or E1, for high affinity interactions with cellular receptors, or for E2 and E1-E2 protein-protein interactions that occur prior to virion:cell membrane fusion. Thus, the present invention provides peptides and methods for treatment and prophylaxis of diseases induced by HCV and related viruses.
The instant invention teaches that HCV envelope glycoprotein E2 has several domains that can be targeted by synthetic peptides to block infection and pathogenesis. The regions of HCV E2 are important for the binding of HCV to its low or high affinity cellular receptors, for rearrangements of E2 or for protein-protein interactions of E2 that occur prior to virion:cell membrane fusion. This invention also teaches and provides synthetic peptides that can inhibit receptor binding and other pre-fusion steps mediated by HCV E2.
Features of hepatitis C virus envelope glycoprotein 2 identified herein provide surprising guidance for the development of vaccines and/or drugs to prevent or treat hepatitis C virus infections. According to one embodiment of the invention, the target for the peptides is E2, the receptor binding protein of HCV. Although proteins, such as soluble CD4, chemokines and antibodies have been developed that block infection by targeting virion receptor binding interactions, peptide mimics of viral surface proteins that block this or other pre-fusion steps have not be described. Prior to the availability of X-ray structural data (Qureshi et al., 1990; Wild, et al., 1993; 1994), several potent HIV-1 inhibitors were developed based on the Gallaher HIV-1 TM fusion protein model (Gallaher et al., 1989). One of these inhibitors, FUZEON® (aka enfuvirtide, DP178; T20) peptide has been shown to substantially reduce HIV-1 load in AIDS patients in clinical trials (Lalezari, et al., 2003). The peptide drugs, which are the subject of the instant invention, were also developed without benefit of X-ray structural data. FUZEON® targets HIV fusion protein and the steps in HIV entry involving fusion between the viral and cellular membrane. Certain hepatitis C virus E2 inhibitory peptides target different steps in the viral replication cycle than are targeted by FUZEON® and other known viral peptide inhibitors. E2-based peptide drugs should be relatively easy to develop, based on our identification of E2 domains that can be targeted by synthetic peptides to block infection and pathogenesis. Once an effective peptide inhibitor is described a non-peptide drug can be developed.
More specifically, the present invention provides for methods of inhibiting viral infection and pathogenesis by hepatitis C virus. The invention is related to the discovery, as described herein, of hepatitis C virus E2 domains that can be targeted by synthetic peptides to block infection and pathogenesis. Various embodiments of the invention provide methods that employ peptides or peptide derivatives to inhibit hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other pre-fusion steps. The present invention provides for methods of treatment and prophylaxis of diseases induced by hepatitis C virus.
Various embodiments of the instant invention provide for pharmaceutical compositions comprising one or more peptides selected from one or more of the following groups.

    
    
        A) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:6;
        B) Peptides homologous to any one of SEQ ID NO:1 to SEQ ID NO:6, except that they are from a different strain of hepatitis C virus.
        C) Peptides homologous to any one of SEQ ID NO:1 to SEQ ID NO:6, except that they are from hepatitis GB virus.
        D) Peptides that are functionally equivalent to any one of SEQ ID NO:1 to SEQ ID NO:6, wherein the functionally equivalent peptide is identical to at least one of SEQ ID NO:1 to SEQ ID NO:6 except that one or more amino acid residues has been substituted with a homologous amino acid, resulting in a functionally silent change, or one or more amino acids has been deleted. An homologous amino acid is an amino acid with chemical or functional similarity to another amino acid. Sets of homologous amino acids are: nonpolar amino acids: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; hydrophobic amino acids: leucine, isoleucine, valine, methionine, alanine, phenylalanine; basic amino acids: lysine, arginine, histidine; acidic amino acids and their amides: aspartic acid, asparginine; glutamic acid, glutamine; aromatic amino acids: tyrosine, tryptophan, phenylalanine, histidine; amino acid alcohols: serine, threonine; and small amino acids: glycine, proline. For example, and not by way of limitation, such peptides may also comprise one or more D-amino acids.
    
    


Various aspects of this embodiment of the invention provide for compositions that comprise one or more peptides having one or more of the following traits:

    
    
        A) Peptides having the amino acid sequence of one or more of SEQ ID NO:1 to SEQ ID NO:42, wherein the N-terminal end of the peptide terminates in an amino group and the C-terminal end of the peptide terminates in a carboxyl group.
        B) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:42, wherein the peptide's N-terminal end does not terminate in an amino group and/or the peptide's C-terminal end does not a terminate in carboxyl group, wherein the peptide's N-terminal end terminates in a moiety selected from the group consisting of: an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group, and/or wherein the peptide's C-terminal end terminates in a moiety selected from the group consisting of an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.
        C) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:42 except that at least one bond linking adjacent amino acid residues is a non-peptide bond.
        D) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:42, except that at least one amino acid residue is in the D-isomer configuration.
        E) Peptides as in groups “A)” or “B)” except that at least one amino acid has been substituted for by a different amino acid (whether a conservative or non-conservative change). Preferably the peptide comprises 1, 2, 3, 4, 5, or more conservative or non-conservative changes. As used herein the term “a conservative change” is preferably defined as substitution in the peptide sequence of an amino acid by a homologous amino acids (for example, a substitution of a leucine for another hydrophobic amino acid such as isoleucine). A non-conservative change is defined as substitution in the peptide sequence of an amino acid by a non-homologous amino acids (for example, a substitution of the acidic aspartic acid for the basic amino acid arginine).
        F) Peptides that are a functional fragment of a peptide as set out in any of groups “A)” to “E)”, above, where the peptides have at least 3 contiguous nucleotides of any one of SEQ ID NO:1 to SEQ ID NO:42. As used herein, the term “a functional fragment of an inhibitory peptide” is preferably defined is a peptide with a shorter sequence consisting of a subset of the amino acids of the inhibitory peptide, which fragment retains the inhibitory properties. For example, “DEFGHKL” could represent a functional fragment of inhibitory peptide “ABCDEFGHIJKLMNOP” if it was inhibitory. And,
        G) Peptides that combine the modification of two or more of part “A”-“F”. For example, a peptide that comprises non-peptide bonds linking two or more of the constituent amino acid residues and has an N-terminal moiety that is not an amino group.
    
    


The instant invention also provides for substantially purified antibodies that specifically react with one or more of the peptides described above.
The instant invention also provides for methods for treating or preventing HCV infections where the method comprises administering one or more of the peptides and/or antibodies as described above.
The instant invention also provides for methods for treating or preventing hepatitis C virus infections where the method comprises administering to a patient in need of such treatment a composition comprising one or more of the peptides and/or antibodies as described above in combination with peptides and/or antibodies or peptides targeting the fusion step mediated at least in part by hepatitis C virus envelope protein 1 as described in international application PCT/US2003/035666 which is herein incorporated by reference. The HCV E2 and E1 peptides and/or antibodies may work synergistically (i.e. they may be active at lower concentrations in combination that when used separately) or they may act in a complimentary or additive fashion.
Abbreviations
HCV—hepatitis C virus
HSA—human serum albumen



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Alignment of protein E2 peptide sequences from two strains of hepatitis C virus showing locations of active peptides. E2 sequences from H77, a genotype 1a strain of HCV (SEQ ID NO:139), and J4, a genotype 1b strain of HCV (SEQ ID NO:140), were aligned. A “:” indicates an identical amino acid and a“.” indicates a chemically similar amino acid in the two sequences. Bars above or below the amino acid sequences indicate the locations of peptides, numbered as in Tables 7 and 8, that inhibit infection by an HCV pseudotype.
FIG. 2. Specificity of HCV E2 inhibitory peptides. E2 peptides, numbered as in Tables 7 and 8, were added to pseudotypes containing the core proteins of HIV and HCV E1, E2, murine leukemia virus surface and transmembrane glycoproteins (SU and TM), or vesicular stomatitis virus glycoprotein (G). Supernatants were also treated with DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2 known to neutralize pseudotype infectivity. Peptide treated and control pseudotypes were added to cells, which were incubated at 37° C. for 72 h. Cell lysates were then tested for luciferase activity as described (Hsu et al., 2003).
FIG. 3: Structure of hepacivirus E2 glycoprotein showing location of active peptides, numbered as in Tables 7 and 8. A two dimensional model of HCV envelope protein 2 (SEQ ID NO:141) was constructed using a proteomics computational tool and comparisons to receptor binding proteins of other RNA viruses. Sequences that resulted in greater than 70% reduction in HCV pseudotype infectivity are indicated.
FIG. 4. Model depicting pre-fusion site of action of HCV E2 peptides. Panel A. HCV E2 peptides disruption of E1-E2 interactions or E2-E2 interactions in HCV virions. Panel B: HCV E2 disruption of HCV virion-receptor interactions.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions for and methods of preventing, treating, or inhibiting Flavivirus infection. The currently disclosed compositions and methods are theorized to operate by inhibiting the fusion between the virion envelope and a cell membrane, the process that delivers the viral genome into the cell cytoplasm.
Various embodiments of the invention, provide the identification and sequence of HCV inhibitory peptides representing specific portions of the HCV E2 glycoprotein. These inhibitory peptides proteins are believed to function by interfering with and blocking receptor binding. These peptides include those represented by SEQ ID NOs 1-42 and derivatives thereof as described below.
In particular aspects of this embodiment of the invention compositions comprising peptides corresponding to the HCV envelope glycoprotein 2 are useful at a broad range of doses (as shown in Example 1 these peptides are effective at inhibiting HCV fusion with the cells).
For purposes of clarity of disclosure, and not by way of limitation, the description of the present invention will be divided into the following subsections:

    (i) Peptides of the invention
    (ii) Utilities of the invention (including compositions and methods for employing the peptides)









TABLE 1







HCV E2 inhibitory peptide 1










PROTEIN
SEQUENCE*












(SEQ ID NO:1)












HCV E2 a
X-LVGLLTPGAKQNIQLINTNGSWHINS-Z









(SEQ ID NO:7)












HCV E2 b
X-FTSLFSSGASQKIQLVNTNGSWHINR-Z









(SEQ ID NO:8)












HCV E2 a
X-LAGLFTSGAKQNIQLINTNGSWHINR-Z









(SEQ ID NO:9)












HCV E2 b
X-FTSFFTRGPSQNLQLVNSNGSWHINS-Z









(SEQ ID NO:10)












HCV E2 a
X-LANLFSSGSKQNLQLINSNGSWHINR-Z









(SEQ ID NO:11)












HCV E2 a
X-LTSFFNPGPQRQLQFVNTNGSWHINS-Z









(SEQ ID NO:12)











HCV E2 a
X-FASLLTPGAKQNIQLINTNGSWHINR-Z

















TABLE 2







HCV E2 inhibitory peptide 2








PROTEIN
SEQUENCE*













HCV E2 1a
X-CNESLNTGWLAGLFYQH-Z
(SEQ ID NO:2)



HCV E2 1b
X-CNDSLHTGFLAALFYTH-Z
(SEQ ID NO:13)


HCV E2 2a
X-CNDSLNTGFIASLFYTY-Z
(SEQ ID NO:14)


HCV E2 3b
X-CNDSLNTGFIAGLFYYH-Z
(SEQ ID NO:15)


HCV E2 4a
X-CNDSLNTGFLASLFYTH-Z
(SEQ ID NO:16)


HCV E2 5a
X-CNDSLQTGFIAGLMYAH-Z
(SEQ ID NO:17)


HCV E2 6a
X-CNDSLQTGFLASLFYTH-Z
(SEQ ID NO:18)
















TABLE 3







HCV E2 inhibitory peptide 3








PROTEIN
SEQUENCE*











(SEQ ID NO:3)










HCV E2 1a
X-YSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGF-Z









(SEQ ID NO:19)










HCV E2 1b
X-YSWGENETDVMLLNNTRPPQGNWFGCTWMNSTGF-Z









(SEQ ID NO:20)










HCV E2 2a
X-YTWGENETDVFILNSTRPPGGSWFGCTWMNSTGF-Z









(SEQ ID NO:21)










HCV E2 3b
X-YRFGVNESDVFLLTSLRPPQGRWFGCVWMNSTGF-Z









(SEQ ID NO:22)










HCV E2 4a
X-YTWGENETDVFLLNSTRPPHGAWFGCVWMNSTGF-Z









(SEQ ID NO:23)










HCV E2 5a
X-YNWGSNETDILLLNNIRPPAGNWFGCTWMNSTGF-Z









(SEQ ID NO:24)









HCV E2 6a
X-YTWGENETDVFMLESLRPPTGGWFGCTWMNSTGF-Z
















TABLE 4







HCV E2 inhibitory peptide 4










PROTEIN
SEQUENCE*












(SEQ ID NO:4)












HCV E2 1a
X-DYPYRLWHYPCTINYTIFKVRMYVGGV-Z









(SEQ ID NO:25)












HCV E2 1b
X-DYPYRLWHYPCTLNFSIFKVRMYVGGV-Z









(SEQ ID NO:26)












HCV E2 2a
X-DYPYRLWHYPCTINYTIFKIRMYVGGV-Z









(SEQ ID NO:27)












HCV E2 3b
X-DYPYRLWHYPCTVNFSIFKVRMFVGGH-Z









(SEQ ID NO:28)












HCV E2 4a
X-DYPYRLWHFPCTANFSVFNIRTFVGGI-Z









(SEQ ID NO:29)












HCV E2 5a
X-HYPYRLWHYPCTVNYTIFKVRMFIGGL-Z









(SEQ ID NO:30)











HCV E2 6a
X-DYAYRLWHYPCTVNFTLHKVRMFVGGT-Z

















TABLE 5







HCV E2 inhibitory peptide 5








PROTEIN
SEQUENCE*











(SEQ ID NO:5)










HCV E2 1a
X-ALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEY-Z









(SEQ ID NO:31)










HCV E2 1b
X-ALSTGLIHLHQNIVDVQYLYGVGSAFVSFAIKWEY-Z









(SEQ ID NO:32)










HCV E2 2a
X-ALSTGLLHLHQNIVDVQYMYGLSPALTKYIVRWEW-Z









(SEQ ID NO:33)










HCV E2 3b
X-RLSTGLIHLHQNIVDVQYLYGVGSAVVGWALKWEF-Z









(SEQ ID NO:34)










HCV E2 4a
X-ALSTGLIHLHQNIVDVQYLYGVGSAVVSWALKWEY-Z









(SEQ ID NO:35)










HCV E2 5a
X-ALSTGLIHLHQNIVDTQYLYGLSSSIVSWAVKWEY-Z









(SEQ ID NO:36)









HCV E2 6a
X-ALSTGLIHLHQNIVDVQYLYGVSTNVTSWVVKWEY-Z
















TABLE 6







HCV E2 inhibitory peptide 6










PROTEIN
SEQUENCE*












(SEQ ID NO:6)












HCV E2 1a
X-VVLLFLLLADARVCSCLWMMLLISQAEA-Z









(SEQ ID NO:37)












HCV E2 1b
X-ILLLFLLLADARVCACLWMMLLIAQAEA-Z









(SEQ ID NO:38)












HCV E2 2a
X-VVLLFLLLADARVCACLWMLILLGQAEA-Z









(SEQ ID NO:39)












HCV E2 3b
X-VVLVFLLLADARVCVALWMMLLISQAEA-Z









(SEQ ID NO:40)












HCV E2 4a
X-VVLAFLLLADARVSAYLWMMFMVSQVEA-Z









(SEQ ID NO:41)












HCV E2 5a
X-IMLVFLLLADARICTCLLILLLICQAEA-Z









(SEQ ID NO:42)











HCV E2 6a
X-IVLMFLVLADARICTCLWLMLLISTVEA-Z







* In tables 1-6 the “X” and “Z” on each peptide respectively represent the N- and C-terminal moiety. As described above the N-terminal moiety may be either an amino group or it may be selected from the group consisting of an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group and/or the peptide's C-terminal moiety may be a carboxy group or it may be a moiety selected from the group consisting of: an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.






Peptides of the Invention
Any peptide or protein which inhibits the fusion between the Hepatitis C virus E2 virion envelope and a cell membrane, including those of Hepatitis C virus E2 which infect human as well as nonhuman hosts, may be used according to the invention. In various embodiments of the invention, these inhibitors may include, but are not limited to peptides related to several membrane-interactive domains of Hepatitis C virus E2 .
Hepatitis C virus E2 inhibitory peptides are, according to the instant invention, identical or homologous to the amino acid sequences
As used herein the term “conservative substitution” preferably refers to substitution in the peptide sequence of an amino acid by a homologous amino acid.
As used herein the term “peptide derivative” preferably refers to: a peptide modified by the addition of one or more groups, including, but not limited to a carbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate
As used herein the term “similar peptides” refers to those peptides having at least 70% identical or chemically similar amino acids. More preferably, it refers to peptides having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or greater identical and/or chemically equivalent amino acid resides.
As used herein the terms “portion thereof” refers to the peptide resulting from the removal of from one or more amino acids from either or both ends of the listed peptide, i.e. a truncated peptide. The number of amino acids removed may vary from 1-10 so long as the remaining fragment is “functional”. As defined herein the term “functional fragment” refers to a fragment capable of inhibiting virus:cell fusion, inhibiting viral infectivity, capable of eliciting an antibody capable of recognizing and specifically binding to non-truncated peptide and/or interfering with hepatitis C virus envelope protein 2-mediated cell infection.
According to the instant invention peptides related to the Hepatitis C virus E2 inhibitory peptides (E2IP) preferably comprise at least three contiguous residues of the E2IP peptides, or a homologous peptide, more preferably they comprise 4, 5, 6, or 7 contiguous residues. Even more preferably they comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more contiguous residues (up to the maximum number of residues in the peptide), and most preferably all residues of these sequences. As used herein the term Hepatitis C virus E2 inhibitory peptides preferably means peptides having a sequence identical to the corresponding portion of the Hepatitis C virus E2 inhibitory protein and peptides in which one or more amino acids are substituted by functionally equivalent amino acids (see infra). The term also refers to derivatives of these peptides, including but not limited to benzylated derivatives, glycosylated derivatives, and peptides which include enantiomers of naturally occurring amino acids. In other embodiments of the invention, the Hepatitis C virus E2 inhibitory peptides, related peptides or derivatives are linked to a carrier molecule such as a protein. Proteins contemplated as being useful according to this embodiment of the invention, include but are not limited to, (human serum albumen). Hepatitis C virus E2 inhibitory peptide-related peptides comprising additional amino acids are also contemplated as useful according to the invention.
Peptides may be produced from naturally occurring or recombinant viral proteins, or may be produced using standard recombinant DNA techniques (e.g. the expression of peptide by a microorganism which contains recombinant nucleic acid molecule encoding the desired peptide, under the control of a suitable transcriptional promoter, and the harvesting of desired peptide from said microorganism). Preferably, the peptides of the invention may be synthesized using any methodology known in the art, including but not limited to, Merrifield solid phase synthesis (Clark-Lewis et al., 1986).
The E2IP, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequences the amino acid sequence hepatitis C virus E2 Inhibitory peptide 1: LVGLLTPGAKQNIQLINTNGSWHINS SEQ ID NO:1; HCV E2 Inhibitory peptide 2 CNESLNTGWLAGLFYQH SEQ ID NO:2; HCV E2 Inhibitory peptide 3, YSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGF SEQ ID NO:3; or HCV E2 Inhibitory peptide 4, DYPYRLWHYPCTINYTIFKVRMYVGGV SEQ ID NO:4; HCV E2 Inhibitory peptide 5, X-ALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEY SEQ ID NO:5; E2 Inhibitory peptide 6, VVLLFLLLADARVCSCLWMMLLISQAEA, SEQ ID NO:6 or a functional portion or functional portions thereof.
Also contemplated are altered sequences analogous to any of SEQ ID NOs 1-42; more preferably analogous to any of SEQ ID NO:1-6 (i.e. altered from any of the sequences referred to herein) in which functionally equivalent amino acid residues are substituted for residues within the sequence, resulting in a functionally silent change. For example, one or more amino acid residues within the sequence can be substituted by replacing the original amino acid with another amino acid, of a similar polarity, that acts as a functional equivalent, resulting in a functionally silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, classes of homologous amino acids are: nonpolar amino acids: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine, polar neutral amino acids: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, hydrophobic amino acids: leucine, isoleucine, valine, methionine, alanine, phenylalanine; basic amino acids: lysine, arginine, histidine; acidic amino acids and their amides: aspartic acid, asparginine; glutamic acid, glutamine; aromatic amino acids, tyrosine, tryptophan, phenylalanine, histidine; amino acid alcohols: serine, threonine and small amino acids: glycine, proline. For example, and not by way of limitation, such peptides may also comprise one or more D-amino acids. Furthermore, in any of the embodiments of the instant invention the peptide may comprise an inefficient carrier protein, or no carrier protein at all.
Further, as noted supra, in any embodiment of the invention the peptide's N-terminal moiety may be either an amino group (as is typically found in naturally occurring proteins/peptides) or it may be selected from the group consisting of an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group and/or the peptide's C-terminal moiety may be a carboxy group (as is typically found in naturally occurring proteins/peptides) or it may be a moiety selected from the group consisting of: an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.
Utility of the Invention
The hepatitis C virus E2 inhibitory peptides of the instant invention may be utilized to inhibit hepatitis C virus infection and may, accordingly, be used in the treatment: of hepatitis C virus infection and also in prophylaxis against hepatitis C virus infection. The peptides of the invention may be administered to patients in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Methods for administering peptides to patients are well known to those of skill in the art; they include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection.
The instant invention provides for compositions, especially pharmaceutical compositions, comprising hepatitis C virus E2 inhibitory peptides, peptide fragments, or derivatives (as described supra) administered via liposomes, microparticles, or microcapsules. Various embodiments of the invention, contemplate the use of such compositions to achieve sustained release of hepatitis C virus E2 inhibitory peptides. Other embodiments contemplate the administration of the FIP or derivatives thereof, linked to a molecular carrier (e.g. HSA).
Various embodiments of the instant invention provide for administration of the hepatitis C virus E2 inhibitory peptides and/or antibodies specific for these peptides to human subjects who suffer from hepatitis C virus infection. In any embodiment the peptides and/or antibodies are typically substantially purified. As used herein the term “substantially purified” refers to a peptide, peptide analog, or antibody that is greater than about 80% pure. More preferably, “substantially purified” refers to a peptide, peptide analog, or antibody that is greater than about 90% or greater than about 95% pure. Most preferably it refers to a peptide, peptide analog, or antibody that is greater than 99% pure. Functionally, “substantially purified” means that it is free from contaminants to a degree that that makes it suitable for the purposes provided herein. Other embodiments provide for the prophylactic administration of the peptides to those at risk for hepatitis C virus infection.
Other embodiments of the instant invention provide for methods for identifying the structure of truncated hepatitis C virus E2 proteins which involved hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other, pre-fusion steps by members of the Flaviviridae family and for the structures themselves.
Other embodiments of the invention provide for a peptide having a formula selected from one or more of the following.
The E2IP, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequences the amino acid sequence hepatitis C virus E2 Inhibitory peptide 1: LVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO:1); HCV E2 Inhibitory peptide 2: CNESLNTGWLAGLFYQH (SEQ ID NO:2); HCV E2 Inhibitory peptide 3: YSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGF (SEQ ID NO:3); or HCV E2 Inhibitory peptide 4: X-DYPYRLWHYPCTINYTIFKVRMYVGGV (SEQ ID NO:4); HCV E2 Inhibitory peptide 5 ALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEY (SEQ ID NO:5); HCV inhibitory peptide 6: VVLLFLLLADARVCSCLWMMLLISQAEA (SEQ ID NO:6); or a functional portion or functional portions thereof of any of these peptides.
According to various embodiments of the instant invention, any of the peptides described herein may comprise an amino group at the amino-terminal end or may be modified to comprise any of the following: an acetyl group, a hydrophobic group or a macromolecular carrier group. Similarly, the carboxy-terminus of any of the peptides may comprise a carboxyl group or may be modified to comprise any of the following groups: an amido group a hydrophobic group or a macromolecular carrier group. In other aspects of this embodiment of the invention, the amino terminal group is a hydrophobic group, a carbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate. In any aspect of this embodiment the carboxy terminal group may be a t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate.
Moreover, aspects of this embodiment also include peptides wherein at least one bond linking adjacent amino acids residues is a non-peptide bond. In particularly preferred aspects of this embodiment the non-peptide bond is an imido, ester, hydrazine, semicarbazoide or azo bond.
Other aspects of this embodiment provide for peptides wherein at least one amino acid is a D-isomer amino acid.
Additional aspects of this embodiment of the invention provide for peptides wherein compromising at least one amino acid substitution has been made so that a first amino acid residue is substituted for a second, different amino acid residue. These substitutions may be conservative or non-conservative, as long as the peptide is still functional according to the instant invention.
Other aspects of this embodiment of the invention provide for peptides wherein at least one amino acid has been deleted. As noted, supra, the peptides according to this embodiment of the invention must comprise at least 3 contiguous amino acids of one of the SEQ ID NOs indicated above and must be a functional segment.
Other embodiments of the invention provide for compositions comprising one or more of the peptides and/or antibodies described herein, either alone, or with a carrier compound. Preferably, the carrier is a pharmaceutically acceptable excipient.
Other embodiments provide for use of the methods described herein in combination with one or more other treatment regimens. For example, one or more peptides of the current invention and/or one or more antibodies specific for peptides of the current invention may be used in combination with or one or more peptides or antibodies targeted to inhibiting the membrane fusion step mediated by hepatitis C virus envelope protein 1. Such peptides are described in international application PCT/US2003/035666 which is herein incorporated by reference in its entirety. The HCV E2 and E1 peptides and/or antibodies may work synergistically (i.e. they may be active at lower concentrations in combination that when used separately) or they may act in a complimentary or additive fashion.
It is noted that any combination of the modifications listed above is considered as part of the instant invention.
EXAMPLES
Example 1
Identification of Hepatitis Virus E2 Peptides that Inhibit Infectivity Mediated by HCV Envelope Proteins
The selective association of a virus with a target cell is usually determined by an interaction between the viral surface glycoproteins and specific receptor or receptors on the cell surface. Receptor-binding is an essential step in the initiation of infection, and precedes other steps such as fusion between the virus and cellular membranes. Virion:receptor interaction(s) can define the host range and cellular or tissue tropism of a virus and may determine pathogenicity. HCV encodes two putative surface glycoproteins, E1 and E2, which are both believed to have carboxyl terminal transmembrane domains that anchor them in the virion envelope. In vitro expression studies have shown that E1 and E2 associate to form heterodimers, which accumulate in the endoplasmic reticulum (ER), the proposed site for HCV assembly and budding (Flint et al., 2004). Several lines of evidence indicate that E2 is the receptor binding protein (Flint and McKeating, 2000). It has been suggested that E1 is the HCV fusion protein (Flint et al., 1999; Garry and Dash, 2003). Other studies, however, indicate that E2, has a class II viral fusion protein structure and represented the fusion protein of HCV (Yagnik et al., 2000), and it is possible that both HCV E1 and E2 have a role in membrane fusion. The lack of in vitro systems for HCV propagation has hampered biological and physiochemical studies on the virion and its mechanism(s) of cell entry, and the cellular receptors remain unknown. HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density lipoprotein receptor (LDLR) to gain entry into cells (Agnello et al., 1999. Truncated soluble versions of E2 have been reported to bind specifically to human cells and were used to identify interactions with CD81 (Pileri et al., 1998; Roccasecca et al., 2003; Cormier et al., 2004), scavenger receptor class B type 1 (SR-B1) (Scarselli et al., 2002), and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) (Pohlmann et al., 2003). The results suggest that E2 may represent a target to develop peptide drugs against hepatitis C virus infection.
Materials And Methods
To overcome the lack of a conventional cell culture system for the propagation of HCV, infectious pseudotype viruses expressing HCV envelope glycoproteins have been generated (Hsu et al., 2003). Pseudotypes with HIV core proteins and HCV envelope proteins were generated by cotransfection of 293-T cells with equal amounts of plasmids expressing HCV E1 and E2 of strain H77 and the HIV envelope-defective proviral genome, pNL4.3.Luc.R−E− (Pohlmann et al., 2003). Peptides from an 18 mer peptide set, overlapping by 7-10 amino acids and representing the entire amino acid sequence of E2 of HCV strain H77 (genotype 1a) and the entire amino acid sequence of HCV strain J4 (genotype 1b) were solubilized in 20% DMSO and diluted (final DMSO concentration <2%). Peptides were incubated at 37° C. with p24 antigen-normalized HCV pseudotype viral supernatants. The average concentration of peptides was  ̃25 μM, however, actual concentrations of some peptides in solution were 10 μM or less due to low solubility in DMSO. Supernatants were also treated with DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2 known to neutralize pseudotype infectivity. Peptide treated and control HCV pseudotypes were added to cells, incubated for 16 hrs, virus removed and cells incubated at 37° C. for 72 h. Cell lysates were then tested for luciferase activity as described (Hsu et al., 2003).
Results and Discussion
Fifty HCV H77 1a E2 peptides were tested in the HCV infectivity assay, and nine demonstrated greater than 70% inhibition of infectivity, with several demonstrating approximately 95% inhibition (Table 7). Of 46 HCV J4 1b E2 peptides tested four demonstrated greater than 70% inhibition of infectivity (Table 8). Several of the inhibitory peptides were overlapping in the E2 sequence, for example HCV H77 1a E2 peptides 4 and 5, 32 and 33, and 43, 44, 45 and 46 (FIG. 1). This result suggests that any of several peptides targets to a particular region may be inhibitory. A comparison of the two sets of E2 peptides, H77 and J4, reveals that while similar peptides can be inhibitory (i.e., peptides 32, 33 and 108, 109), while other peptides with closely related sequences are not inhibitory (i.e., peptides 4, 5, and 99).
Selected E2 inhibitory peptides were added to pseudotypes containing the core proteins of HIV and murine leukemia, virus surface, and transmembrane glycoproteins (MuLV SU and TM), or vesicular stomatitis virus glycoprotein (VSV G). These peptides inhibited pseudotypes with HCV E1 and E2 (FIG. 2A), but failed to significantly inhibit the MuLV or VSV pseudotypes (FIGS. 2B and 2C). The results indicate that these HCV E2 inhibitory peptides are specific for inhibition of the infectivity of HCV. These results also demonstrate the potential of E2 peptides as antiHCV drugs.







TABLE 7







Identification of HCV E2 inhibitory peptides (strain H77)


that inhibit HCV infectivity.









HCV H77 1a




peptide #
Luciferase units†
Percent inhibition












 1
250,376
44.1


 2
447,336
0.2


 3
447,906
0.05


 4
10,620
97.6


 5
48,000
89.3


 6
503,446
−12.3


 7
381,340
14.9


 8
113,650
74.6


 9
501,126
−11.8


10
334,196
25.4


11
360,410
19.6


12
417,706
6.8


13
313,323
31.1


14
279,626
37.6


15
253,410
43.5


16
403,430
10.0


17
254,516
43.2


18
435,026
2.9


19
301,406
32.7


20
231,373
48.4


21
242,223
45.9


22
245,900
45.2


23
367,916
17.9


24
391,886
19.7


25
480,280
7.2


26
216,706
51.6


27
575,206
−28.4


28
394,780
11.9


29
297,353
33.6


30
655,040
−46.2


31
419,263
6.4


32
85,086
81.0


33
22,406
95.0


34
354,696
21.8


35
153,553
66.7


36
535,016
−19.5


37
585,553
−30.7


38
345,110
23.0


39
400,756
11.6


40
442,346
1.3


41
434,743
3.0


42
353,516
19.1


43
32,283
92.8


44
91,266
79.6


45
24,703
94.5


46
103,040
77.0


47
195,320
56.4


48
290,786
35.1


49
307,310
31.4


50
58,790
87.9


VIRUS ALONE
448,123


Virus plus anti-
10,309
97.6


E2 2/69a


Virus plus anti-
3,567
99.2


E2 9/27





†The numbers represent the number of luciferase units (lumens) produced after infection by either the HCV or the MLV pseudotype in the presence of the peptide at a concentration of ~25 μM. Results above 70% inhibition are indicated in bold-face type.













TABLE 8







Identification of HCV E2 inhibitory peptides (strain J4)


that inhibit HCV infectivity.









HCV J4 1b




peptide #
Luciferase units†
Percent inhibition












54
372,393
17.9


81
480,623
−7.3


82
173,156
61.4


83
518,993
−15.8


84
392,023
12.5


85
112,260
74.9


51
237,086
47.1


86
398,110
11.2


87
399,700
10.8


88
412,776
7.9


89
449,293
−0.3


90
423,326
5.5


91
160,883
69.1


92
372,400
16.9


93
409,220
9.7


94
311,736
30.4


95
538,110
−20.1


96
544,596
−21.5


97
218,673
51.2


98
467,636
−4.4


99
111,043
75.2


100
518,190
−15.6


101
502,096
−12.0


102
377,216
15.8


103
305,690
31.8


104
419,876
6.3


105
552,170
−23.2


106
193,533
60.4


107
402,976
11.1


108
40,853
90.9


109
96,893
79.4


110
602,506
−34.5


111
632,613
−39.2


112
527,950
−17.8


113
570,553
−27.3


114
270,190
39.7


115
475,713
−6.2


116
394,096
12.1


117
359,236
19.8


119
69,220
84.6


120
463,243
−3.4


121
338,200
24.5





†The numbers represent the number of luciferase units (lumens) produced after infection by either the HCV or the MLV pseudotype in the presence of the peptide at a concentration of ~25 μM. Samples were compared to controls described in Table 7. Results above 70% inhibition are indicated in bold-face type.













TABLE 9







Sequence and location of peptides shown in Table 7.











HCV 1a






H77


peptide
Peptide

HCV E2 IP


#
location*
Amino acid Sequence
overlap















1
379-396
AGVDAETHVTGGSAGRTT
(SEQ ID NO 43)




2
386-403
HVTGGSAGRTTAGLVGLL
(SEQ ID NO 44)


3
393-410
GRTTAGLVGLLTPGAKQN
(SEQ ID NO 45)
HCV E2IP 1


4
399-417
VGLLTPGAKQNIQLINTN
(SEQ ID NO 46)
HCV E2IP 1


5
407-424
AKQNIQLINTNGSWHINS
(SEQ ID NO 47)
HCV E2IP 1


6
414-431
INTNGSWHINSTALNCNE
(SEQ ID NO 48)
HCV E2IP 1


7
421-438
HINSTALNCNESLNTGWL
(SEQ ID NO 49)
HCV E2IP 1/2


8
428-445
NCNESLNTGWLAGLFYQH
(SEQ ID NO 50)
HCV E2IP 2


9
442-459
FYQHKFNSSGCPERLASC
(SEQ ID NO 51)
HCV E2IP 2


10
449-466
SSGCPERLASCRRLTDFA
(SEQ ID NO 52)


11
456-473
LASCRRLTDFAQGWGPIS
(SEQ ID NO 53)


12
463-480
TDFAQGWGPISYANGSGL
(SEQ ID NO 54)


13
470-487
GPISYANGSGLDERPYCW
(SEQ ID NO 55)


14
477-494
GSGLDERPYCWHYPPRPC
(SEQ ID NO 56)


15
486-501
PYCWHYPPRPCGIVPAKS
(SEQ ID NO 57)


16
491-508
PRPCGIVPAKSVCGPVYC
(SEQ ID NO 58)


17
501-515
PAKSVCGPVYCFTPSPVV
(SEQ ID NO 59)


18
505-522
PVYCFTPSPVVVGTTDRS
(SEQ ID NO 60)


19
512-529
SPVVVGTTDRSGAPTYSW
(SEQ ID NO 61)
HCV E2IP 3


20
526-543
TYSWGANDTDVFVLNNTR
(SEQ ID NO 62)
HCV E2IP 3


21
533-550
DTDVFVLNNTRPPLGNWF
(SEQ ID NO 63)
HCV E2IP 3


22
540-557
NNTRPPLGNWFGCTWMNS
(SEQ ID NO 64)
HCV E2IP 3


23
547-564
GNWFGCTWMNSTGFTKVC
(SEQ ID NO 65)
HCV E2IP 3


24
554-571
WMNSTGFTKVCGAPPCVI
(SEQ ID NO 66)
HCV E2IP 3


25
561-578
TKVCGAPPCVIGGVGNNT
(SEQ ID NO 67)


26
568-585
PCVIGGVGNNTLLCPTDC
(SEQ ID NO 68)


27
575-592
GNNTLLCPTDCFRKHPEA
(SEQ ID NO 69)


28
582-599
PTDCFRKHPEATYSRCGS
(SEQ ID NO 70)


29
589-606
HPEATYSRCGSGPWITPR
(SEQ ID NO 71)


30
596-613
RCGSGPWITPRCMVDYPY
(SEQ ID NO 72)
HCV E2IP 4


31
603-620
ITPRCMVDYPYRLWHYPC
(SEQ ID NO 73)
HCV E2IP 4


32
610-627
DYPYRLWHYPCTINYTIF
(SEQ ID NO 74)
HCV E2IP 4


33
617-634
HYPCTINYTIFKVRMYVG
(SEQ ID NO 75)
HCV E2IP 4


34
624-641
YTIFKVRMYVGGVEHRLE
(SEQ ID NO 76)
HCV E2IP 4


35
631-648
MYVGGVEHRLEAACNWTR
(SEQ ID NO 77)
HCV E2IP 4


36
638-655
HRLEAACNWTRGERCDLE
(SEQ ID NO 78)


37
645-662
NWTRGERCDLEDRDRSEL
(SEQ ID NO 79)


38
652-669
CDLEDRDRSELSPLLLST
(SEQ ID NO 80)


39
659-676
RSELSPLLLSTTQWQVLP
(SEQ ID NO 81)


40
666-683
LLSTTQWQVLPCSFTTLP
(SEQ ID NO 82)


41
673-690
QVLPCSFTTLPALSTGLI
(SEQ ID NO 83)
HCV E2IP 5


42
680-697
TTLPALSTGLIHLHQNIV
(SEQ ID NO 84)
HCV E2IP 5


43
687-704
TGLIHLHQNIVDVQYLYG
(SEQ ID NO 85)
HCV E2IP 5


44
694-711
QNIVDVQYLYGVGSSIAS
(SEQ ID NO 86)
HCV E2IP 5


45
701-718
YLYGVGSSIASWAIKWEY
(SEQ ID NO 87)
HCV E2IP 5


46
708-725
SIASWAIKWEYVVLLFLL
(SEQ ID NO 88)
HCV E2IP 5/6


47
715-732
KWEYVVLLFLLLADARVC
(SEQ ID NO 89)
HCV E2IP 5/6


48
722-739
LFLLLADARVCSCLWMML
(SEQ ID NO 90)
HCV E2IP 6


49
729-746
ARVCSCLWMMLLISQAEA
(SEQ ID NO 91)
HCV E2IP 6


50
756-773
WMMLLISQAEAALENLVI
(SEQ ID NO 92)
HCV E2IP 6





*Numbering refers to the numbering provided at Genbank Accession NP_671491













TABLE 10







Sequence and location of peptides shown in Table 8.










HCV





1b J4


peptide
Peptide

HCV E2 IP


#
location*
Amino acid sequence
overlap















54
(379-396)
AGVDGETHTTGRVAGHTT
(SEQ ID NO 93)




80
(386-403)
HTTGRVAGHTTSGFTSLF
(SEQ ID NO 94)
HCV E2IP 1


81
(393-410)
GHTTSGFTSLFSSGASQK
(SEQ ID NO 95)
HCV E2IP 1


82
(400-417)
TSLFSSGASQKIQLVNTN
(SEQ ID NO 96)
HCV E2IP 1


83
(407-424)
ASQKIQLVNTNGSWHINR
(SEQ ID NO 97)
HCV E2IP 1


84
(421-438)
HINRTALNCNDSLQTGFF
(SEQ ID NO 98)
HCV E2IP 1/2


85
(428-445)
NCNDSLQTGFFAALFYAH
(SEQ ID NO 99)
HCV E2IP 2


51
(435-452)
TGFFAALFYAHKFNSSGC
(SEQ ID NO 100)
HCV E2IP 2


86
(442-459)
FYAHKFNSSGCPERMASC
(SEQ ID NO 101)
HCV E2IP 2


87
(449-466)
SSGCPERMASCRPIDWFA
(SEQ ID NO 102)


88
(456-473)
MASCRPIDWFAQGWGPIT
(SEQ ID NO 103)


89
(463-480)
DWFAQGWGPITYTKPNSS
(SEQ ID NO 104)


90
(477-494)
PNSSDQRPYCWHYAPRPC
(SEQ ID NO 105)


91
(484-501)
PYCWHYAPRPCGVVPASQ
(SEQ ID NO 106)


92
(491-508)
PRPCGVVPASQVCGPVYC
(SEQ ID NO 107)


93
(498-515)
PASQVCGPVYCFTPSPVV
(SEQ ID NO 108)


94
(505-522)
PVYCFTPSPVVVGTTDRS
(SEQ ID NO 109)


95
(512-529)
SPVVVGTTDRSGVPTYSW
(SEQ ID NO 110)
HCV E2IP 3


96
(519-536)
TDRSGVPTYSWGENETDV
(SEQ ID NO 111)
HCV E2IP 3


97
(526-543)
TYSWGENETDVMLLNNTR
(SEQ ID NO 112)
HCV E2IP 3


98
(533-550)
ETDVMLLNNTRPPQGNWF
(SEQ ID NO 113)
HCV E2IP 3


99
(540-557)
NNTRPPQGNWFGCTWMNS
(SEQ ID NO 114)
HCV E2IP 3


100
(554-571)
WMNSTGFTKTCGGPPCNI
(SEQ ID NO 115)
HCV E2IP 3


101
(561-578)
TKTCGGPPCNIGGVGNRT
(SEQ ID NO 116)


102
(568-585)
PCNIGGVGNRTLICPTDC
(SEQ ID NO 117)


103
(575-592)
GNRILICPTDCFRKHPEA
(SEQ ID NO 118)


104
(582-599)
PTDCFRKHPEATYTKCGS
(SEQ ID NO 119)


105
(589-606)
HPEATYTKCGSGPWLTPR
(SEQ ID NO 120)


106
(596-613)
KCGSGPWLTPRCLVDYPY
(SEQ ID NO 121)
HCV E2IP 4


107
(603-620)
LTPRCLVDYPYRLWHYPC
(SEQ ID NO 122)
HCV E2IP 4


108
(610-627)
DYPYRLWHYPCTLNFSIF
(SEQ ID NO 123)
HCV E2IP 4


109
(617-634)
HYPCTLNFSIFKVRMYVG
(SEQ ID NO 124)
HCV E2IP 4


110
(631-648)
MYVGGVEHRLNAACNWTR
(SEQ ID NO 125)
HCV E2IP 4


111
(638-655)
HRLNAACNWTRGERCNLE
(SEQ ID NO 126)


112
(645-662)
NWTRGERCNLEDRDRSEL
(SEQ ID NO 127)


113
(652-669)
CNLEDRDRSELSPLLLST
(SEQ ID NO 128)


114
(659-676)
RSELSPLLLSTTEWQILP
(SEQ ID NO 129)


115
(666-683)
LLSTTEWQILPCAFTTLP
(SEQ ID NO 130)


116
(673-690)
QILPCAFTTLPALSTGLI
(SEQ ID NO 131)
HCV E2IP 5


117
(680-697)
TTLPALSTGLIHLHQNIV
(SEQ ID NO 132)
HCV E2IP 5


118
(694-711)
QNIVDVQYLYGVGSAFVS
(SEQ ID NO 133)
HCV E2IP 5


119
(708-725)
AFVSFAIKWEYILLLFLL
(SEQ ID NO 134)
HCV E2IP 5/6


120
(722-739)
LFLLLADARVCACLWMML
(SEQ ID NO 135)
HCV E2IP 6


121
(729-746)
ARVCACLWMMLLIAQAEA
(SEQ ID NO 136)
HCV E2IP 6





*Numbering refers to the numbering provided at Genbank Accession BAA01583






The peptides of Tables 7-10 are an overlapping set of peptides representing E2 of two strains of HCV (H77 and J4). SEQ ID NO's 1-6 are longer versions of the “hits” highlighted in yellow in Tables 7-10. The rationale for the design of SEQ ID NOs:1-6 is that the optimum inhibitory peptide may be include flanking sequences), and that the ultimate optimum peptide may be a fragment of this longer peptide. SEQ ID NOs:7-42 are variants of SEQ ID NOs:1-6 representing analogous sequences from the E2 proteins of the other major genotypes of HCV.
Example 2
Proteomic Computational Model of HCV E2
Because HCV cannot be propagated in cell culture, insufficient numbers of virions are available to perform structural analyses. Thus, the molecular structure of HCV E2 has not been determined and is currently unknown. In the absence of an X-ray crystallographic structure of HCV E2, it is possible to derive useful structural information using newly developed computational analyses supplemented by comparisons to other viral glycoproteins of known structure. Such a model of HCV E2 can be useful in defining the potential mechanisms of action of E2 inhibitory peptides.
Materials and Methods
Representatives of the most common subtypes of hepatitis C virus were used for sequence and structural comparisons. The strains examined a human prototype HCV strain H77 (subtype 1a, Genbank Accession NP—751921), strain HC-J4 (subtype 1b, Genbank Accession BAA01583), strain NDM59 (subtype 2a, Genbank Accession AF169005), strain TrKj (subtypes 3b, Genbank Accession D49374), strain ED43 (subtype 4a, Genbank Accession Y11604), strain EUH1480 (subtype 5a , Genbank Accession Y13184), strain euhk2 (subtype 6a, Genbank Accession Y12083).
Methods to derive general models of surface glycoproteins have been described previously (Gallaher et al., 1989). PRSS3, a program derived from rdf2 (Pearson and Lipman, 1988), which uses the Smith-Waterman sequence alignment algorithm (Smith and Waterman, 1981), was used to determine the significance of protein alignments. PRSS3 is part of the FASTA package of sequence analysis programs available by anonymous ftp from ftp.virginia.edu. Default settings for PRSS3 were used, including the blosum50 scoring matrix, gap opening penalty of 12, and gap extension penalty of 2. MacMolly (Soft Gene GmbH, Berlin) was used to locate areas of limited sequence similarity and to perform Chou-Fasman and Robson-Gamier analyses (Biou et al., 1988; Chou and Fasman, 1974). PHDsec (Columbia University Bioinformatics Center, http://cubic.bioc.columbia.edu/predictprotein/) was the preferred method of secondary structure prediction (Rost and Liu, 2003). PHDsec predicts secondary structure from multiple sequence alignments by a system of neural networks, and is rated at an expected average accuracy of 72% for three states, helix, strand and loop. Domains with significant propensity to form transmembrane helices were identified with TMpred (ExPASy, Swiss Institute of Bioinformatics, http://www.ch.embnet.org/software/TMPRED_form.html). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins (Hofmann and Stoffel, 1993). Sequences with a propensity to partition into the lipid bilayer were identified with Membrane Protein eXplorer version 2.2a from the Stephen White laboratory using default settings (White et al., 2003).
Results and Discussion
A two-dimensional model of HCV E2 was developed based on the application of proteomics computational analyses and comparison to the known structures of other receptor-binding viral envelope proteins (FIG. 3). The HCV E2 secondary structure structures drawn in FIG. 3 conform to the PHDsec secondary structure alignment algorithm and are also generally consistent with both Chou-Fasman and Robson-Garnier predictions. An important feature of the E2 model is the predominance of beta sheet structures in the amino terminal two-thirds of the molecule and alpha helical structures in the carboxyl terminal third of the molecule. Dicysteine linkages were predicted on the basis of comparison to the envelope glycoproteins of retroviruses. In retroviral envelope proteins adjacent cysteines that are separated by greater than 15 amino acids are typically covalently bonded to each other. Clusters of four cysteines that are each separated by fewer than 15 amino acids are typically covalently bonded to a nonadjacient cysteine in the cluster. The first two long predicted alpha helices in the carboxyl terminal third of E2 form a predicted stem structure analogous to the stem region of other flavivirus envelope proteins (Allison et al., 1999). The depicted transmembrane domain structure was predicted by the TMPRED algorithm.
HCV E2 inhibitory peptides map to regions on the HCV E2 model that correspond to an amino terminal region, a cysteine cluster, the stem and transmembrane domain (shaded regions in FIG. 3). These domains of HCV E2 can be involved in hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other pre-fusion steps. HCV E2 inhibitory peptides can be used to interfere with these early steps in HCV infection as depicted in FIG. 4.
The present invention is not to be to be construed as limited in scope by the specific embodiments described herein. Rather, the specific embodiments described are only illustrative. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of each of which is incorporated by reference in its entirety. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention
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## Claims

1. A peptide consisting of a fragment of SEQ ID NO:5 that comprises the sequence of SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87.

2. A peptide consisting of SEQ ID NO:5.

3. The peptide of claim 1, wherein the fragment comprises SEQ ID NO:85.

4. The peptide of claim 3, wherein the fragment consists of SEQ ID NO:85.

5. The peptide of claim 1, wherein the fragment comprises SEQ ID NO:86.

6. The peptide of claim 5, wherein the fragment consists of SEQ ID NO:86.

7. The peptide of claim 1, wherein the fragment comprises SEQ ID NO:87.

8. The peptide of claim 7, wherein the fragment consists of SEQ ID NO:87.

9. A pharmaceutical composition comprising a peptide, wherein the peptide consists of SEQ ID NO:5 or a fragment of SEQ ID NO:5 comprising SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87, wherein the peptide optionally has:
a) in place of an N-terminal amino group hydrogen, an N-terminal moiety that is an acetyl group, a hydrophobic group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group;
b) in place of the C-terminal carboxylate group hydroxyl, a C-terminal moiety that is an amino group, a hydrophobic group, a t-butyloxycarbonyl group or a macromolecular carrier group; or
c) an amino acid residue that is in the D-isomer configuration.

10. The composition of claim 9, wherein the peptide has:
a) in place of an N-terminal amino group hydrogen, an N-terminal moiety that is an acetyl group, a hydrophobic group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group; or
b) in place of the C-terminal carboxylate group hydroxyl, a C-terminal moiety that is a hydrophobic group, a t-butyloxycarbonyl group or a macromolecular carrier group.

11. The composition of claim 10, wherein either the N-terminal moiety or the C-terminal moiety of the peptide is a macromolecular carrier group selected from a lipid conjugate, polyethylene glycol, or a carbohydrate.

12. The composition of claim 9, wherein the peptide has an amino acid that is in a D-isomer configuration.

13. A method of treating or preventing hepatitis C virus (HCV) infection in a patient, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition according to any one of claims 9-12.

14. The method of claim 13, further comprising administering to the patient an antibody produced using as an immunogen a peptide, wherein the peptide immunogen consists of SEQ ID NO:5 or a fragment of SEQ ID NO:5 comprising SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87.